Ribosomal binding of modified tRNA anticodons related to thermal stability.
The physicochemical contributions of modified nucleosides to tRNA functions are not well understood. In order to determine the contributions of specific modifications to tRNA stability as well as to ribosomal binding, ten variously modified yeast tRNA(Phe) anticodon stems and loops (tRNA(Phe)AC) were synthesized. Thermal denaturation studies on these synthetic 17mers show dramatic stabilization (or destabilization) by the presence of the various naturally occurring nucleoside modifications. Adapting a novel molecular biology approach (initially pioneered by Moazed and Noller), the interactions of these variously modified anticodons with the E. coli 16S rRNA "P-site" residues are being quantitated. The binding (affinity) constant (kD) of the tRNA(Phe)AC to the 8 of the ten 16S rRNA nucleosides that interact with tRNA and synthetic anticodons are being examined. We postulate that the "stabilizing" modifications (m1G37, psi 39, and m5C40) in the presence of an "open loop" will dramatically increase the binding affinity of the tRNA(Phe)AC to the 30S E. coli ribosomal subunit when compared to unmodified tRNA(Phe)AC. On the other hand, "destabilizing" modifications are expected to reduce the binding affinity of the tRNA(Phe)AC to the E. coli 30S ribosomal subunit. The results from these experiments have demonstrated the importance of nucleoside modifications to tRNA stability and ribosomal binding affinity, and will relate the structural contributions of nucleoside modifications to tRNA function.
Ashraf
S S
SS
Department of Biochemistry, North Carolina State University, Raleigh 27695, USA.
Guenther
R
R
Ye
W
W
Lee
Y
Y
Malkiewicz
A
A
Agris
P F
PF
D000926:Anticodon / Q000378:metabolism*; D004926:Escherichia coli; D006358:Hot Temperature; D009690:Nucleic Acid Conformation; D012329:RNA, Bacterial / Q000378:metabolism; D012331:RNA, Fungal / Q000737:chemistry / Q000378:metabolism*; D012336:RNA, Ribosomal, 16S / Q000378:metabolism; D012360:RNA, Transfer, Phe / Q000737:chemistry / Q000378:metabolism*; D012270:Ribosomes / Q000378:metabolism*; D012441:Saccharomyces cerevisiae; D017423:Sequence Analysis, RNA
D000926:Anticodon; D012329:RNA, Bacterial; D012331:RNA, Fungal; D012336:RNA, Ribosomal, 16S; D012360:RNA, Transfer, Phe